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Protective Role of Bacillithiol in Superoxide Stress and Fe–S Metabolism in Bacillus Subtilis

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Protective Role of Bacillithiol in Superoxide Stress and Fe–S Metabolism in Bacillus Subtilis ORIGINAL RESEARCH Protective role of bacillithiol in superoxide stress and Fe–S metabolism in Bacillus subtilis Zhong Fang & Patricia C. Dos Santos Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27016. Keywords Abstract Bacillithiol, dehydratase, Fe–S cluster, Suf, superoxide stress. Glutathione (GSH) serves as the prime thiol in most organisms as its depletion increases antibiotic and metal toxicity, impairs oxidative stress responses, and Correspondence affects Fe and Fe–S cluster metabolism. Many gram-positive bacteria lack GSH, Patricia C. Dos Santos, Department of but instead produce other structurally unrelated yet functionally equivalent thi- Chemistry, Wake Forest University, Winston- ols. Among those, bacillithiol (BSH) has been recently identified in several low Salem, NC 27016. Tel: 336-758-3144; Fax: G+C gram-positive bacteria. In this work, we have explored the link between 336-758-4656; E-mail: [email protected] BSH and Fe–S metabolism in Bacillus subtilis. We have identified that B. subtilis lacking BSH is more sensitive to oxidative stress (paraquat), and metal toxicity Funding Information (Cu(I) and Cd(II)), but not H2O2. Furthermore, a slow growth phenotype of This research was funded by the National BSH null strain in minimal medium was observed, which could be recovered Science Foundation (MCB-1054623). upon the addition of selected amino acids (Leu/Ile and Glu/Gln), supplementa- tion of iron, or chemical complementation with BSH disulfide (BSSB) to the Received: 12 March 2015; Revised: 16 April growth medium. Interestingly, Fe–S cluster containing isopropylmalate isomer- 2015; Accepted: 17 April 2015 ase (LeuCD) and glutamate synthase (GOGAT) showed decreased activities in BSH null strain. Deficiency of BSH also resulted in decreased levels of intracel- MicrobiologyOpen 2015; 4(4): 616–631 lular Fe accompanied by increased levels of manganese and altered expression – doi: 10.1002/mbo3.267 levels of Fe S cluster biosynthetic SUF components. Together, this study is the first to establish a link between BSH and Fe–S metabolism in B. subtilis. Introduction in its reduced form and its disulfide form (GSSG) with the cellular ratio of GSH:GSSG estimated to be around Biothiols are involved in numerous cellular functions, 100:1 (Fahey et al. 1978; Meister and Anderson 1983) as including but not limited to a variety of biosynthetic opposed to cysteine, whose thiol:disulfide ratio lies pathways, detoxification by conjugation, metal metabo- around 25:1 (Newton et al. 2009; Sharma et al. 2013). lism, and cell division (Winterbourn and Metodiewa Early studies have shown that GSH plays a key role in a 1999; Jacob et al. 2006; Hider and Kong 2011). They can wide array of biochemical functions. First, GSH can be classified as large molecular weight thiols, also called directly serve as an antioxidant by donating electrons to protein thiols, and low molecular weight (LMW) free thi- reactive radicals through autoxidation reactions to form ols (Sen and Packer 2000). Cysteine is the most common GSSG (Meister and Anderson 1983; Meister 1988; Ches- LMW biothiol and its sulfhydryl group provides unique ney et al. 1996). Thus, under oxidative stress conditions, chemical functionality distinct from other standard amino the GSH/GSSG ratio may fall into the 1–10 range (Reed acids. Its neutral pKa, nucleophilicity, redox properties, 1990). Second, GSH serves as the substrate of biochemical metal ion affinity, and bonding characteristics make Cys a reactions. For example, dehydroascorbate reductase which versatile site for many chemical processes (Bulaj et al. is a key component of the GSH-ascorbate cycle uses GSH 1998; Jacob et al. 2006; Roos et al. 2013). as a substrate to produce ascorbate to detoxify H2O2 In the late 19th century, glutathione (GSH) was identi- (Crook 1941). Besides the various roles of GSH on detox- fied as another abundant LMW thiol widely distributed ifying oxidants and toxins, GSH is also involved in in most cells (Rey-Pailhade 1888a,b). GSH mainly exists chemical modification of protein cysteine residues 616 ª 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Z. Fang & P. C. Dos Santos Protective Role of Bacillithiol (Dalle-Donne et al. 2009). S-glutathionylation is consid- mycothiol exerted a small but significant protective effect ered a specific posttranslational modification on protein against WhiD cluster loss under oxidative stress (Crack cysteine residues in response to oxidative stress or nitro- et al. 2009). sative stress (Giustarini et al. 2005; Martinez-Ruiz and In some low-(G+C)-content gram-positive bacteria, Lamas 2007; Dalle-Donne et al. 2009). Several findings such as Bacillus species, bacillithiol (BSH) is a major have also reported the vital role for S-glutathionylation in LMW thiol recently discovered in bacteria lacking GSH redox-regulation, energy metabolism, cellular signaling, and MSH (Newton et al. 2009). Because of the crucial calcium homeostasis, protein folding, and stability (Reed roles of GSH and MSH in thiol-redox homeostasis, pro- 1990; Cotgreave et al. 2002; Dalle-Donne et al. 2003, tein posttranslational modification, and xenobiotic 2009; Pan and Berk 2007; Demasi et al. 2008; Rodriguez- detoxification, it is anticipated that some of these func- Pascual et al. 2008). tions might also be performed by BSH (Helmann 2011). Interestingly, the effect of GSH on the superoxide- The BSH redox potential was characterized to be sensitive Fe–S cluster-containing aconitase (ACN) of À221 mV through in vitro coupled assay with GSH/ Escherichia coli has also been demonstrated (Gardner GSSG, which is higher than Cys (À223 mV) and Co- and Fridovich 1993a). The results showed a ~20% ASH (À234 mV) (Sharma et al. 2013). This implies that decrease in ACN activity in a GSH-deficient E. coli BSH does not have better capacity to buffer oxidative strain, displaying a phenotype which could be recovered stress through autoxidation reaction. However, consider- À upon addition of GSH to the growth medium (Gardner ing the low thiol pKa value of BSH (pKaSH/S = 7.97) and Fridovich 1993a). Earlier research indicated that and the relatively high concentration of BSH (0.5– À the reactivation of O2 -inactivated ACN in cell extract 5 mmol/L) (Sharma et al. 2013), it is still possible that was blocked by adding ethylenediaminetetraacetic acid BSH is preferable to Cys and CoA as a major redox buf- (EDTA) or 2,20-dipyridyl (DP) (Kennedy et al. 1983; fer in B. subtilis. In addition, BSH has been shown to Gardner and Fridovich 1992). This observation led to modulate the intracellular labile zinc pool by forming a À the proposal that the reactivation of O2 -inactivated tetrahedral BSH2:zinc complex with two sulfur and two ACN would involve the restoration of [3Fe–4S] clusters oxygen ligands (Ma et al. 2014). Although BSH is not by free Fe2+. Overall, in this model, GSH would act as an essential metabolite under standard laboratory condi- either a reductant or Fe2+ donor to facilitate repair of tions, initial studies have identified that bacterial strains damaged Fe–S clusters. Recently, it has been reported unable to synthesize this thiol displayed impaired sporu- that GSH also acts as a key component of the cytoplas- lation, sensitivity to acid and salt, depleted NADPH mic labile iron pool providing further support for the level, and increased sensitivity to fosfomycin, hypochlo- important role of GSH in iron homeostasis (Hider and rite, diamide, and methylglyoxal (Gaballa et al. 2010; Kong 2011). Moreover, changes in the redox ratio of Lamers et al. 2012; Roberts et al. 2013; Chandrangsu GSH/GSSG has been correlated with induction of man- et al. 2014; Posada et al. 2014). While defects in metab- ganese (Mn)-dependent superoxide dismutase expression olism resulting from BSH phenotypes have been identi- (Gardner and Fridovich 1993b). Despite compelling evi- fied, biochemical pathways involving BSH have not been dence for GSH’s involvement in oxidative stress, thiol fully explored. homeostasis, and Fe–S cluster metabolism, the complete The goal of this work was to investigate the role of inventory of biological processes involving GSH as well BSH in Fe–S metabolism under normal and stress con- as the exact mechanism of GSH-dependent reactions is ditions. We show that lack of BSH caused a slow still not fully understood. growth phenotype in minimal medium, which may be Although GSH is recognized as the dominant LMW due to the lower activity of Fe–S enzymes involved in thiol in eukaryotes and gram-negative prokaryotes, GSH branched chain amino acid biosynthesis. Furthermore, is not detected in many gram-positive species. This growth assays under various stress conditions revealed a observation suggested the existence of other LMW thiols protective role of BSH against copper stress, superoxide replacing the role of GSH in those species. In fact, in stress, cadmium stress, and iron starvation. Along with Actinobacteria, mycothiol (MSH) was identified as a the parallel Fe–S enzyme assays, thiol analysis, and novel cysteine derivative (Newton et al. 1996). Mycobac- intracellular metal analysis suggest the involvement of terium smegmatis strains lacking mycothiol are more BSH in superoxide stress and metal homeostasis. More- sensitive to reactive oxygen and nitrogen species, such as over, western blot analysis was performed on Fe–S hydrogen peroxide, menadione, plumbagin, and t-butyl cluster biosynthetic proteins in wild-type and mutant hydrogen peroxide (Newton et al. 1999, 2008; Rawat strains. Metal analysis of a BSH deletion strain under et al. 2002). A recent study on WhiD, a member of the paraquat (PQ) stress also revealed a link between BSH WhiB-like family of Fe–S proteins, indicated that methyl and Mn homeostasis. ª 2015 The Authors.
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